We analyzed 31 polymorphisms in 18 candidate genes within the SOF cohort to identify genetic risk factors for osteoporosis. It was of particular interest that women with the ALOX15_G48924T T/T genotype had a 33% higher rate of hip fracture in this study. This polymorphism was the only one with an allelic association with hip fracture in this study. ALOX15 and ALOX12 are contiguous genes located within the 17p13 region of the human genome, which contains a quantitative trait locus that affects BMD in the hip, spine [29
], and wrist [30
]. However, previous studies have found inconsistent associations between SNPs in ALOX15 and BMD or fracture data [31
]. The ALOX15_G48924T SNP is located within the 5′ flanking region (−272 bp) of ALOX15. This polymorphism is of interest because a C-to-T substitution at ALOX15 position −292 was shown to create a novel transcription factor binding site for SPI1 [35
]. SPI1 selectively binds to the −292 T allele, and transcription assays in primary human macrophages showed that −292 C/T heterozygous individuals expressed three times more ALOX15 mRNA than −292 C/C individuals [35
]. Higher ALOX15 mRNA levels were also observed in monocytes from heterozygous −292 C/T carriers [36
]. The ALOX15_G48924T SNP (G-272T) examined herein may be in linkage disequilibrium with the functional C-292T polymorphism, leading to differential ALOX15 expression and increased risk of fracture for the variant allele. Alternately, G-272T may itself be functional. Consistent with our findings in the female SOF cohort, there were significant associations between SNPs within the 5′ flanking region of ALOX15 and BMD in Japanese women in two different studies [32
]. By contrast, two studies did not observe associations between 5′-flanking ALOX15 SNPs and BMD in Chinese women [33
] or BMD and fracture in postmenopausal white women [31
]. Taken together, the results of genetic association studies performed in this and two other female populations indicate that genetic variation within the 5′ promoter region of ALOX15 may contribute to osteoporosis-related traits. Measuring associations between 5′-flanking ALOX15 SNPs and BMD and fracture in emerging genome-wide association study data sets may help confirm these associations.
Prolactin is a peptide hormone that when present at high levels is associated with decreased levels of estrogen and testosterone. Prolactin may also have direct effects on osteoblast function and bone formation [37
]. In this study, women carrying one or two copies of the PRL_T228C C allele had a ~20% lower rate of nonvertebral and nonhip fractures. The function of the intronic T228C polymorphisms is currently unknown. High prolactin levels have been associated with osteopenia, decreased bone density, and increased osteoporosis risk, possibly as a result of a reduction in estrogen levels [40
]. In addition, long-term administration of raloxifene, which has been shown to decrease fracture risk in postmenopausal women with osteoporosis, decreases serum prolactin levels [42
Bone morphogenetic protein 2 is a growth factor belonging to the transforming growth factor beta super-family that plays a role in osteoblast differentiation. The gene for bone morphogenetic protein 2 (BMP2) was identified as an osteoporosis candidate locus by genome-wide linkage mapping in human populations [43
]. To date, two BMP2 SNPs have been associated with fracture [43
] and BMD [44
]; however, the associations are not consistent [43
]. In the present study, women with the intronic BMP2_A125611G G/G genotype had a 51% higher risk of vertebral fracture. The function of the BMP2_A125611G polymorphism is unknown, and it is possible that this SNP is functional or in linkage disequilibrium with a functional variant.
MMP-2 is a determinant of bone remodeling and mineralization and plays a crucial role in forming and maintaining the osteocytic canalicular network [46
]. Serum concentrations of MMP-2 have been related to markers of bone turnover including bone alkaline phosphatase, osteocalcin, and cross-linked N-telopeptides of type I collagen [47
]. A previous study also found that serumMMP-2 levels may also increase with increasing bone turnover [48
]. In the present study, women with one copy of the MMP2_C595T T allele (located in the 5′ promoter at position −1586) had a 21% lower adjusted rate of vertebral fracture, and women with two copies had a 56% lower adjusted rate.
Several studies have investigated the association between estrogen receptor alpha (ESR1) gene variants and osteoporosis [49
]. In the present study, neither the Pvu
II (rs2234693) nor the Xba
I (rs9340799) polymorphisms were associated with hip or nonvertebral/nonhip fracture risk. Women with the ESR1_C1335G G/G genotype had a 64% higher rate of vertebral fracture, compared with women with the C/C genotype. The ESR1_C1335G variant codes for a synonymous P325P substitution in exon 4. A previous study of late postmenopausal women found that mean femoral neck BMD, but not lumbar spine BMD, was significantly lower in the homozygous G/G women compared with the homozygous C/C women [52
]. Another study found that after 6 months of treatment with raloxifene, subjects with C/C or C/G genotype of P325P mutation had significantly lower total cholesterol and low-density lipoprotein cholesterol concentrations, and higher decreases of total cholesterol when compared with those with the G/G genotype [53
]. Our results and those of Jurada et al. [52
] suggest that the codon 325 G/G genotype is associated with increased risk of vertebral fracture and lower femoral neck BMD.
There were no significant genetic associations with total hip BMD. The strongest association with BMD was found for the CYP1A1_A6570G polymorphism with a 4%increase in BMD for the G/G genotype compared with the A/A genotype. The inconsistency of genetic associations with fracture andBMDand across fracture types is consistent with studies in mice, which indicate that there are skeletal-site-specific genetic loci for bone mass and strength [54
]. Previous findings have demonstrated that a wide array of skeletal phenotypes were polygenic with complex segregation patterns [57
]. Beamer et al. [55
] showed that several quantitative trait loci were responsible for both femoral and vertebral measures of BMD, whereas other quantitative trait loci were unique to femurs or vertebrae. Unique genetic factors contributing to trabecular and cortical bone mass have also been identified [54
]. Another possibility is that the fracture findings are possibly spurious as a result of the multiple comparisons that were made.
Although this study found several positive genetic associations with osteoporotic outcomes, most of the investigated polymorphisms were not associated with fracture risk or BMD, and none of the previously unreported polymorphisms were consistently significantly associated with multiple fracture types or BMD sites. Previously reported associations between polymorphisms in COL1A1 [58
], LRP5 [65
], CASR [72
], CALCR [73
], and MTHFR [74
] andBMDor fracture were not replicated in this study. Inconsistencies between this and previous studies may be due in part to differences in study size, specific SNPs assayed, sex-specific effects, ethnic background, and menopausal status, all of which influence genetic associations with BMD and fracture risk. Several previous studies [58
] had small sample sizes (<300 participants), which may have led to spurious associations. For several genes, we examined different SNPs than those previously reported [63
]. Two of the previous studies only found associations in men [65
] or premenopausal women [72
]. Several studies were conducted on nonwhite participants [67
This study is limited in making conclusions regarding whether the examined genes play an important role in fracture risk or BMD because of the limited number of polymorphisms per gene studied in this population. Even though the selected polymorphisms based on prior investigation seemed to be promising, no single SNP can explain the variation of an entire gene. Although the SOF cohort is a well-characterized and appropriate cohort to use for osteoporosis- related studies, particularly within the population of elderly white women, results from a single population likely cannot be generalized to all possible populations. Finally, interactions between environmental factors and other genes may have obscured important subgroup associations with the candidate gene polymorphisms.
In the past decades, several approaches have been attempted to identify osteoporosis genes; however, the genes contributing to osteoporosis risk remain poorly defined. As with most complex diseases, it is generally assumed that many gene variants are responsible, with each contributing a subtle effect. Inconsistent results may be due to a lack of statistical power to detect the subtle effects of the responsible gene variants, a lack of standardized methods and approaches to identify the variants, or the selection of the wrong candidate genes. Recently the consortium approach to genetic studies, as exemplified for osteoporosis by the “genetic markers for osteoporosis” (GENOMOS) consortium [51
], has remedied some of the most important pitfalls of candidate gene studies by standardizing phenotypes and genotypes, increasing sample sizes, improving power, and reducing false discovery rates. In addition, replication has become well established as the gold standard in genetic association studies to overcome problems with multiple testing and false-positive discoveries. The increasing use of genome-wide screening approaches, which exacerbate the discovery of false-positive findings, requires well-conducted replication studies in a variety of populations to confirm true novel genetic associations and increase generalizability of findings to more than one population. Making genotype data available from phenotypically well-characterized individual studies (such as those reported here) not only provides an opportunity for future confirmation of genome-wide association study results for specific genes, but also contributes to future meta-analyses. In addition, disclosure of negative as well as positive associations is essential to minimize the risk of publication bias. Ioannidis [8
] argues that the large majority of molecular epidemiology results should be null and that scientific journals should publish all studies with null results, provided study limitations are acknowledged. Rebbeck et al. [85
] provide a framework for prioritizing the publication of reports that are likely to provide more meaningful information about disease etiology.